Potential of cell retention techniques for large‐scale high‐density perfusion culture of suspended mammalian cells

Abstract

This review focuses on cultivation of mammalian cells in a suspended perfusion mode. The major technological limitation in the scaling-up of these systems is the need for robust retention devices to enable perfusion of medium as needed. For this, cell retention techniques available to date are presented, namely, cross-flow filters, hollow fibers, controlled-shear filters, vortex-flow filters, spin-filters, gravity settlers, centrifuges, acoustic settlers, and hydrocyclones. These retention techniques are compared and evaluated for their respective advantages and potential for large-scale utilization in the context of industrial manufacturing processes. This analysis shows certain techniques have a limited range of perfusion rate where they can be implemented (most microfiltration techniques). On the other hand, techniques were identified that have shown high perfusion capacity (centrifuges and spin-filters), or have a good potential for scale-up (acoustic settlers and inclined settlers). The literature clearly shows that reasonable solutions exist to develop large-scale perfusion processes.

Separation of CHO cells using hydrocyclones

“…Previous studies have shown that some cell lines are susceptible to hydrodynamic stress, presenting a significant decrease in cell viability when subject to stress levels above a threshold limit (Born et al 1992). However, due to the low viability drops observed in this work, the viability of CHO cells separated by hydrocyclones was kept above 92%, which is higher than the cell viability obtained for most cell retention devices reported by Voisard et al (2003). In this review article, a compilation of 21 works using different cell retention devices showed that in only two cases cell viability was kept at values higher than the minimum viability (92%) obtained in the present work.…”

“…In CHO-cell perfusion cultures with spin-filters, the efficiencies attained were in the range of 75-95% (Iding et al 2000), as compared to a minimum efficiency of 97.9% found for hydrocyclones in the present work. Furthermore, unlike hydrocyclones, fouling and clogging of spin-filter meshes is considered a major problem, which may cause premature interruption of the culture process (Esclade et al 1991;Deo et al 1996;Voisard et al 2003). Furthermore, when compared to other hydrocyclone geometries, the efficiencies found in the present work are higher than the best efficiency (81%) obtained for HeLa cells by Lübberstedt et al (2000b).…”

“…Centrifuges may suffer from cell adhesion and clogging, besides their high mechanical complexity and high cost (Jäger 1992;Tokashiki et al 1990). Sedimentation-based apparatuses are susceptible to cell adhesion, and the high residence time needed to perform the separation exposes the cells to non-controlled environments (Searles et al 1994;Voisard et al 2003). Thus, special operation strategies like back-pulsing, to prevent cell build-up at the drain ports, and refrigeration systems, to induce cell aggregation and so enhance particle sedimentation, have to be employed (Lipscomb et al 2004).…”

Stable hybridoma cultivation in a pilot‐scale acoustic perfusion system: Long‐term process performance and effect of recirculation rate

“…Different cell-retention systems are available for mammalian-cell culture and have been extensively discussed recently (Castilho and Medronho, 2002;Voisard et al, 2003;Woodside et al, 1998). Cell-retention systems available for perfusion systems at relatively large-scale (100-1,000 L/d) are spinfilters (Deo et al, 1996;Yabannavar et al, 1994), hollow fibers/alternating tangential-flow filters (Voisard et al, 2003), centrifuges (Johnson et al, 1996;Takamatsu et al, 1996), settlers (Arai et al, 1993), and acoustic cell separators (Gorenflo et al, 2002).…”

“…Cell-retention systems available for perfusion systems at relatively large-scale (100-1,000 L/d) are spinfilters (Deo et al, 1996;Yabannavar et al, 1994), hollow fibers/alternating tangential-flow filters (Voisard et al, 2003), centrifuges (Johnson et al, 1996;Takamatsu et al, 1996), settlers (Arai et al, 1993), and acoustic cell separators (Gorenflo et al, 2002). Acoustic separators have no physical barrier and no moving mechanical parts and are, therefore, less prone to fouling and mechanical failure.…”